A new method using simple solid phase extraction for the rapid gas-chromatographic mass-spectrometric determination of 11-dehydro-thromboxane B2 in urine

A new gas-chromatographic mass-spectrometric method for the rapid determination of 11-dehydro-thromboxane B2 in urine, the major metabolite of systemic thromboxane formation, has been developed. Excellent sample clean-up was obtained in a single step by adsorption of 11-dehydro-TXB2 on phenylboronate cartridges, vigorous polar and organic washing and elution with an acidic methanol mixture. Then the pentafluorobenzylester trimethylsilylether derivative of 11-dehydro-TXB2 was formed and quantified in isotope dilution technique by negative chemical ionisation gas-chromatography tandem mass-spectrometry. The daughter fragments m/z 243/247 of the parent ion m/z 511/515 were monitored. Recovery was linear and quantitative over a wide range, accuracy was 95 + 7% and precision was 11% down to the very low pg range in biologic samples. Independent validation of this very fast extraction method with a reference method applying extensive sample purification with consecutive reversed and straight phase extraction, precolumn derivatisation, reversed phase high pressure liquid chromatography and tandem gas-chromatography mass-spectrometry gave excellent agreement of values. Values for 11-dehydro-TXB2 excretion in 8 healthy controls were 501 + 298 (range 231 to 1141) ng/g creatinin. Excretion was suppressed by aspirin, moderately elevated in heavy smokers (range 680 to 1540) and increased in patients with venous thrombosis or pulmonary embolism (2370 to 13350 ng/g creatinin). This rapid extraction method is useful for the highly specific and sensitive determination of 11-dehydro-TXB2 in large sample numbers.     

Identification of 11-dehydro-TXB2 as a suitable parameter for monitoring thromboxane production in the human

In order to identify suitable parameters for measurement of thromboxane production the metabolism of TXB₂ was studied in the human. [³H₈]-TXB₂ was given intravenously to a healthy human volunteer. Blood samples were collected for 50 min after the injection, and urine was collected for 24 hours. The urinary and blood metabolic profiles were visualized by the use of two-dimensional TLC and autoradiography. Identification of metabolites was achieved with GC/MS and in some cases by cochromatography with reference compounds in TLC and GC.In blood, unmetabolized TXB₂ was the dominating compound during the first 30 min. Three less polar metabolites appeared, two of which were identified as 11-dehydro-TXB₂ and 11, 15-didehydro-13, 14-dihydro-TXB₂, respectively. The third compound was tentatively identified as 15-dehydro-13, 14-dihydro-TXB₂.Since 11-dehydro-TXB₂ was one of the major metabolites in blood as well as urine, it was deemed suitable as target for measurement of thromboxane production . The advantages of 11-dehydro-TXB₂ over its parent compound, TXB₂, were demonstrated in experiments where unlabeled TXB₂ was injected i.v. to a human volunteer, and the blood and urinary levels of both compounds were then followed by radioimmunoassay. Measured levels of 11-dehydro-TXB₂ were found to give a more reliable picture of metabolic events than TXB₂, the latter compound to a large extent reflecting technical difficulties during blood sample collection.     

Circulating and urinary thromboxane B2 metabolites in the rabbit: 11-dehydro-thromboxane B2 as parameter of thromboxane production

The metabolism of thromboxane B₂ was studied in the rabbit. The aim of the study was to identify metabolites in blood and urine that might serve as parameters for monitoring thromboxane production in vivo. [5, 6, 7, 8, 9, 11, 12, 14, 15-³H₈]-Thromboxane B₂ was administered by i.v. injection to rabbits, and blood samples and urine were collected with brief intervals. The metabolic profiles were visualized by two-dimensional thin layer chromatography and autoradiography, and the structures of five major metabolites were determined using chromatographic and mass spectrometric methods.In urine the major metabolites were identified as 11-dehydro-TXB₂ and 2, 3, 4, 5-tetranor-TXB₁, and other prominent products were 11-dehydro-2, 3, 4, 5-tetranor-TXB₁, 2, 3-dinor-TXB₁ and 2, 3-dinor-TXB₂. In the circulation, TXB₂ was found to disappear rapidly. The first major metabolite to appear was 11-dehydro-TXB₂, which also remained a prominent product in blood for the remainder of the experiment (90 min). With time, the profile of circulating products became closely similar to that in urine. TXB₂ was not converted into 11-dehydro-TXB₂ by blood cells or plasma. The dehydrogenase catalyzing its formation was tissue bound and was found to have a widespread occurrence: the highest conversion was found in lung, kidney, stomach and liver.The results of the present study suggest that 11-dehydro-TXB₂ maybe a suitable parameter for monitoring thromboxane production in vivo in the rabbit in blood as well as urinary samples, and possibly also several tissues. This was also demonstrated in comparative studies using radioimmunoassays for TXB₂ and 11-dehydro-TXB₂.     

Determination of celecoxib in human plasma and rat microdialysis samples by liquid chromatography tandem mass spectrometry

Methods for the determination of celecoxib in human plasma and rat microdialysis samples using liquid chromatography tandem mass spectrometry are described. Celecoxib and an internal standard were extracted from plasma by solid-phase extraction with C₁₈ cartridges. Thereafter compounds were separated on a short narrow bore RP C₁₈ column (30×2 mm). Microdialysis samples did not require extraction and were injected directly using a narrow bore RP C₁₈ column (70×2 mm). The detection was by a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface. The compounds were detected in the negative ion mode using the mass transitions m/z 380→316 and m/z 366→302 for celecoxib and internal standard, respectively. The assay was validated for human plasma over a concentration range of 0.25–250 ng/ml using 0.2 ml of sample. The assay for microdialysis samples (50 μl) was validated over a concentration range of 0.5–20 ng/ml. The method was utilised to determine pharmacokinetics of celecoxib in human plasma and in rat spinal cord perfusate.     

Determination and pharmacokinetic study of chiisanogenin in rat plasma by ultra performance liquid chromatography-tandem mass spectrometry

Introduction – Chiisanogenin existing in many Acanthopanax species has been reported to possess anti‐inflammatory, antibacterial and antiplatelet aggregatory activities. Objective – To develop and validate a rapid and sensitive ultra performance liquid chromatography‐tandem mass spectrometry method for the determination of chiisanogenin in rat plasma and to investigate its pharmacokinetics after oral administration of chiisanogenin or the extract of Acanthopanax sessiliflorus fruits. Methodology – The sample pretreatment involved a one‐step extraction of 0.2 mL plasma with diethyl ether. Acetaminophen was used as the internal standard. The separation was carried out on an ACQUITY UPLC? BEH C₁₈ column with a mobile phase of acetonitrile‐5 mM ammonium acetate (90:10, v/v) at a flow rate of 0.2 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. Results – A high sample throughput was achieved with an analysis time of 1.1 min per sample. The calibration curve was linear (r² ≥ 0.99) over the concentration range of 5–500 ng/mL with a lower limit of quantification (LLOQ) of 5 ng/mL. The intra‐day and inter‐day precision (relative standard deviation, R.S.D.) values were below 11% and the accuracy (relative error, R.E.) was within 8% at all three quality control (QC) levels. Conclusion – The method was successfully applied to the pharmacokinetic study of chiisanogenin in rat after oral administration of chiisanogenin and the extract of Acanthopanax sessiliflorus fruits. Other constituents in the extract affected the pharmacokinetic behavior of chiisanogenin. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantification of cidofovir in human serum by LC-MS/MS for children

A new method for the quantification of cidofovir (CDV), an acyclic nucleotide analogue of cytosine with antiviral activity against a broad-spectrum of DNA viruses, in human serum, using high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been developed. A strong anion exchange (SAX) solid-phase extraction procedure was applied for the sample preparation. The tandem mass spectrometer was tuned in the multiple reaction monitoring mode to monitor the m/z 278.1-->234.9 and the m/z 288.1-->133.1 transitions for CDV and the internal standard 9-(2-phosphonylmethoxyethyl)guanine (PMEG), respectively, using negative electrospray ionization. The MS/MS response was linear over the concentration range from 78.125 ng/ml to 10,000 ng/ml, with a lower limit of quantification of 78.125 ng/ml. The intra- and inter-day precisions (relative standard deviation (%)) for CDV were less than 7.8% and the accuracies (% of deviation from nominal level) were within +/-12.1% for quality controls. The novel LC-MS/MS method allowed a specific, sensitive and reliable determination of CDV in human serum and was applied to investigate the yet unknown pharmacokinetic properties of CDV in a paediatric cancer patient.     

Simultaneous quantitation of dexamethasone palmitate and dexamethasone in human plasma by liquid chromatography/tandem mass spectrometry

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for simultaneous quantitation of dexamethasone palmitate and dexamethasone in human plasma was developed. After sample preparation by protein precipitation and liquid-liquid extraction, the analytes and internal standard (IS) were separated on a Venusil XBP-C8 column using gradient elution. Multiple reaction monitoring of dexamethasone palmitate, dexamethasone and IS used the precursor to product ion transitions at m/z 631.8-->373.1, m/z 393.2-->147.1 and m/z 264.2-->58.1, respectively. The method was linear over the ranges 1.5-1000ng/mL for dexamethasone palmitate and 2.5-250ng/mL for dexamethasone with intra- and inter-day precisions of <10% and accuracies of 100+/-7%. The assay was applied to a clinical pharmacokinetic study involving the injection of dexamethasone palmitate to healthy volunteers.     

Simultaneous determination of flupirtine and its major active metabolite in human plasma by liquid chromatography–tandem mass spectrometry

A rapid, selective and sensitive HPLC–tandem mass spectrometry method was developed and validated for simultaneous determination of flupirtine and its active metabolite D-13223 in human plasma. The analytes and internal standard diphenhydramine were extracted from plasma samples by liquid–liquid extraction, and chromatographed on a C₁₈ column. The mobile phase consisted of acetonitrile–water–formic acid (60:40:1, v/v/v), at a flow rate of 0.5 ml/min. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI). The method has a limit of quantitation of 10 ng/ml for flupirtine and 2 ng/ml for D-13223, using 0.5-ml plasma sample. The linear calibration curves were obtained in the concentration range of 10.0–1500.0 ng/ml for flupirtine and 2.0–300.0 ng/ml for D-13223. The intra- and inter-run precision (RSD), calculated from quality control (QC) samples was less than 7.2% for flupirtine and D-13223. The accuracy as determined from QC samples was less than 5% for the analytes. The overall extraction recoveries of flupirtine and D-13223 were determined to be about 66% and 78% on average, respectively. The method was applied for the evaluation of the pharmacokinetics of flupirtine and active metabolite D-13223 in volunteers following peroral administration.     

Determination of 3-methoxy-4-hydroxyphenylethylene glycol in urine using reversed-phase liquid chromatography with column switching and electrochemical detection

A column-switching method was developed for the determination of total 3-methoxy-4-hydroxy-phenylethyleneglycol (MHPG) in urine. This was performed by first treating samples with β-glucuronidase, followed by extraction with ethyl acetate. The reconstituted extracts with injected onto an HPLC system containing an amperometric detector and tandem Nucleosil C₁₈ and C₈ reversed-phase columns connected by a switching valve. The total analysis time for MHPG was 12 min. The limit of detection was 0.18 ng, or 9 μg/l for 20-μl injections of a 1.0-ml reconstituted extract prepared from 1.0 ml of urine. The linear range extended up to 80 mg/l. The within-day precision for a urine sample containing 170 μg/l total MHPG was ±6% and the day-to-day precision was ±15%. The average levels determined by this method for total MHPG in normal subjects showed good agreement with previous literature values. This approach could be modified for the determination of free MHPG by using only ethyl acetate extraction for sample pretreatment.     

96-Well liquid-liquid extraction liquid chromatography-tandem mass spectrometry method for the quantitative determination of ABT-578 in human blood samples

We report here a quantitative method for the analysis of ABT-578 in human whole blood samples. Sample preparation was achieved by a semi-automated 96-well format liquid-liquid extraction (LLE) method. Aluminum/polypropylene heat seal foil was used to enclose each well of the 96-well plate for the liquid-liquid extraction. A liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) method with pre-column regeneration was developed for the analysis of sample extracts. Selective reaction monitoring (SRM) of the mass transitions m/z 983-935 and m/z 931-883 was employed for the detection of ABT-578 and internal standard, respectively. The ammonium adduct ions [M + NH(4)](+) generated from electrospray ionization were monitored as the precursor ions. The assay was validated for a linear dynamic range of 0.20-200.75ng/ml. The correlation coefficient (r) was between 0.9959 and 0.9971. The intra-assay CV (%) was between 1.9 and 13.5% and the inter-assay CV (%) was between 4.7 and 11.3%. The inter-assay mean accuracy was between 86.4 and 102.5% of the theoretical concentrations.     

Determinants of platelet activation in hypertensives with microalbuminuria

Microalbuminuria is a predictor of adverse outcome in hypertension. We evaluated in vivo platelet activation, by urinary 11-dehydrothromboxane (TX)B₂ and plasma P-selectin, in hypertensives with or without microalbuminuria, and its possible association with oxidative stress, by urinary 8-iso-prostaglandin (PG)F_2α and endothelial dysfunction. Sixty essential hypertensive patients, with (n = 30) or without (n = 30) microalbuminuria, and 30 controls were studied. Endothelial function was assessed by nitric oxide products, intercellular adhesion molecule (ICAM)-1, and asymmetric dimethylarginine (ADMA) levels. Urinary 11-dehydro-TXB₂ excretion was higher in microalbuminuric (median 805 pg/mg creatinine) compared to nonmicroalbuminuric patients or controls (414 and 291 pg/mg, respectively; P < 0.0001). Plasma P-selectin was significantly higher in patients with microalbuminuria (median 136 ng/ml) as compared to those without microalbuminuria or controls (85 and 65 ng/ml; P < 0.0001). Urinary 8-iso-PGF_2α excretion was also enhanced in microalbuminuric (median 279 pg/mg creatinine) compared to nonmicroalbuminuric patients or controls (157 and 146 pg/mg, respectively; P < 0.0001). A significant impairment in endothelial function was found in microalbuminuric patients, with decreased nitric oxide and increased ICAM-1 and ADMA levels. Multivariate regression analysis showed that urinary 8-iso-PGF_2α excretion (beta = 0.49; P < 0.0001) and microalbuminuria (beta = 0.36; P < 0.001) were independently related to 11-dehydro-TXB₂ in hypertensives. Vitamin E supplementation (900 mg daily for 1 month) in 10 hypertensives with microalbuminuria was associated with normalization in median 11-dehydro-TXB₂ and 8-iso-PGF_2α. We conclude that lipid peroxidation is a major determinant of persistent platelet activation in hypertensive patients with microalbuminuria.     

Sensitive and rapid liquid chromatography/tandem mass spectrometric assay for the quantification of chloroquine in dog plasma

A simple, sensitive and rapid liquid chromatography/tandem mass spectrometric (LC-MS/MS) method was developed and validated for quantification of chloroquine, an antimalarial drug, in plasma using its structural analogue, piperazine bis chloroquinoline as internal standard (IS). The method is based on simple protein precipitation with methanol followed by a rapid isocratic elution with 10 mM ammonium acetate buffer/methanol (25/75, v/v, pH 4.6) on Chromolith SpeedROD RP-18e reversed phase chromatographic column and subsequent analysis by mass spectrometry in the multiple reaction monitoring mode (MRM). The precursor to product ion transitions of m/z 320.3-->247.2 and m/z 409.1-->205.2 were used to measure the analyte and the IS, respectively. The assay exhibited a linear dynamic range of 2.0-489.1 ng/mL for chloroquine in dog plasma. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.4 and 2.0 ng/mL, respectively in 0.05 mL plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range of 2.0-489.1 ng/mL. A run time of 2.0 min for a sample made it possible to achieve a throughput of more than 400 plasma samples analyzed per day. The validated method was successfully used to analyze samples of dog plasma during non-clinical study of chloroquine.     

Determination of the GABAB receptor agonist CGP 44532 (3-amino-2-hydroxypropylmethylphosphinic acid) in rat plasma after pre-column derivatization by micro-high-performance liquid chromatography combined with negative electrospray tandem mass spectrometry

An assay, based on pre-column derivatization and micro-high-performance liquid chromatography–tandem mass spectrometry, was developed for the determination of the GABA_B agonist CGP 44532 in rat plasma. CGP 44532, a highly polar 3-amino-2(S)-hydroxypropylmethylphosphinic acid, presented difficulties in developing a chromatographic method for the analysis of the compound in rat plasma. Instead of analyzing the target compound directly, it was derivatized prior to separation to a 4-nitrobenzylcarbamate isopropyliden derivative. In order to reach the required quantitation limit, on-line solid-phase extraction was utilized for sample clean-up and reversed-phase micro-column high-performance liquid chromatography, for separation of the plasma samples. The separated compounds were detected by negative electrospray tandem mass spectrometry in selected reaction monitoring mode. The derivatives show good chromatographic and mass spectrometric properties and both the target compound and the internal standard, could be eluted as symmetrical peaks with good signal/noise ratio. The MS–MS detection was selective and sensitive due to the straight fragmentation pattern. After injection of 200-μl sample aliquots, the limit of quantification was 10 ng ml⁻¹. The analytical assay is useable in the range of 10–500 ng ml⁻¹.     

Quantitative determination of nystatin in human plasma using LC-MS after inhalative administration in healthy subjects

The antifungal polyene antibiotics nystatin was tested in a clinical trial to describe pharmacokinetics and safety after repeated administration of Nystatin "Lederle" sterile powder in healthy volunteers. To monitor the nystatin concentration-time profile in plasma we developed a sensitive method in the range of 1-100ng/ml based on liquid chromatography coupled with tandem mass spectrometry. The target substance was separated from the biological matrix on C(18) solid-phase extraction cartridges with methanol. The Chromatography was performed isocratically using a reversed phase Caltrex Resorcinearene column. The mobile phase consisted of 5mM ammonium formate buffer and acetonitrile (40:60, v/v). The mass spectrometer works with electrospray ionization in its positive selected ion monitoring (SIM) mode using the respective MH(+) ions, m/z 926.6 for nystatin and m/z 924.4 for amphotericin B as internal standard. The method validation was performed according to the demands and international criteria for validation of bioanalytical methods and was successfully applied to the quantification of nystatin in human plasma in the pharmacokinetic trial.     

Rapid and sensitive LC–MS/MS assay for the quantitation of 20(S)-protopanaxadiol in human plasma

This paper describes a rapid and sensitive method for the quantitation of 20(S)-protopanaxadiol (PPD) in human plasma based on high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analyte and internal standard (I.S.), ginsenoside Rh₂, were extracted from plasma by liquid–liquid extraction and separated on a Zorbax extend C₁₈ analytical column using methanol–acetonitrile-10 mM ammonium acetate (47.5:47.5:5, v/v/v) as mobile phase. Detection was by tandem mass spectrometry using electrospray ionization in the positive ion mode and multiple reaction monitoring (MRM). The assay was linear over the concentration range 0.1–100.0 ng/ml with a limit of detection of 0.05 ng/ml. The method was successfully applied to a clinical pharmacokinetic study in healthy volunteers after a single oral administration of a PPD 25 mg capsule.     

Optimisation of an extraction method for the determination of prostaglandin E2 in plasma using experimental design and liquid chromatography tandem mass spectrometry

A new extraction method has been developed for the extraction of prostaglandin E(2) (PGE(2)) from human plasma of patients suffering chronic inflammatory disorders. The extraction solvents were optimised systematically and simultaneously by using a central composite design. The optimised method involves precipitation of the protein fraction, centrifugation, evaporation and dissolution of the supernatant in the mobile phase, screening to confirm the presence of the analyte, and quantification of the positive samples by liquid chromatography tandem ion-trap mass spectrometry. Tandem mass spectrometry in negative mode was performed by isolating and fragmenting the ion [PGE(2)-H](-) signal m/z 351. Identification and quantification was carried out by extracting the ion fragment chromatograms at 333, 315 and 271 m/z. The quantitative determination was linear for the low nanogram (1-50 ng/ml) and upper picogram (400-1000 pg/ml) range studied, using 15 and 0.5 ng/ml of internal standard, respectively. The lower limit of detection was 2.5 pg for an injection volume of 25 microl. The optimised extraction method showed high reproducibility (coefficients of variation<4%) and recovery values, estimated from standard addition experiments, ranging from 96 to 98%.     

A capillary liquid chromatographic/tandem mass spectrometric method for the quantification of gamma-aminobutyric acid in human plasma and cerebrospinal fluid

A sensitive and reliable method for the determination of gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter, in human plasma and cerebrospinal fluid (CSF) has been developed. The method is based on capillary liquid chromatography (LC)/tandem mass spectrometry (MS/MS) using deuterium-labeled GABA (gamma-aminobutyric acid-2,2-D(2), GABA-d(2)) as internal standard. Pre-column derivatization with 7-fluoro-4-nitrobenzoxadiazole (NBD-F) was deployed, allowing both effective in-line pre-concentration and sensitive tandem MS detection of the analyte. An extraction column (10 mm x 0.25 mm, 7 microm, C(18)) was used for preconcentrating and stacking the sample. Separation was carried out on an analytical column (50 mm x 0.25 mm, 5 microm, C(18)). Characteristic precursor-to-product ion transitions, m/z 267--> 249 (for NBD-GABA) and m/z 269--> 251 (for NBD-GABA-d(2)) were monitored for the quantification. A linear calibration curve from 10 to 250 ng/mL GABA with an r(2) value of 0.9994 was obtained. Detection limit was estimated to be 5.00 ng/mL GABA (S/N = 3). Human plasma and CSF samples were analyzed. The concentrations of GABA were found to be 98.6 +/- 33.9 ng/mL (mean +/- S.D., n = 12), and 44.3 +/- 10.0 ng/mL (n = 6) in plasma and CSF, respectively.     

Pulmonary metabolism of prostacyclin (PGI2) in the rabbit.

Metabolism of prostacyclin, [9-³H]PGI₂, was examined in the isolated perfused rabbit lung and the post-microsomal supernate of rabbit lung homogenate. Two major metabolites of [9-³H]PGI₂ from the lung perfusate were separated by thin-layer chromatography and radiometric gas-chromatography. These two products were identified as 6 keto-PGF_1α and 6,15 diketo-13,14 dihydro PGF_1α by mass-spectrometry; they represented 65% and 14% of the total radioactivity. When [9-³H]PGI₂ was incubated with the lung homogenate in the presence of either NAD⁺ or NADP⁺, more than 36% and 25%, respectively, was converted to the 6,15 diketo-13,14 dihydro metabolite.     

High-performance liquid chromatographic method for the simultaneous determination of the camptothecin derivative irinotecan hydrochloride, CPT-11, and its metabolites SN-38 and SN-38 glucuronide in rat plasma with a fully automated on-line solid-phase extraction system, PROSPEKT

We established a high-performance liquid chromatography (HPLC) method for the simultaneous determination of the camptothecin (CPT) derivative, irinotecan hydrochloride (CPT-11) and its metabolites, 7-ethyl-10-hydroxycamptothecin (SN-38) and SN-38 glucuronide (SN-38G) in rat plasma with a fully automated on-line solid-phase extraction system, PROSPEKT. Plasma samples were pretreated with 0.146 M H₃PO₄ to inactivate carboxylesterase and β-glucuronidase in rat plasma, and added with the internal standard solution (0.146 M H₃PO₄ containing 1 μg/ml CPT) and then analyzed. The method was validated for CPT-11 (5 to 25 000 ng/ml), SN-38 (5 to 2500 ng/ml) and SN-38G (2.5 to 500 ng/ml). This method enabled the determination of many samples within a relatively short time with easy sample preparation. It also had four advantages compared with conventional determination methods, i.e. automation of a complicated sample preparation, time-saving by the simultaneous determination of three compounds, the direct determination of SN-38G, and the small amount of plasma required for the determination.